Recent advances in the field of targeted modifications of a genome have made is so that routine targeted modifications may soon be possible. Significant advances have been made in the last few years towards the development of methods and compositions to target and cleave genomic DNA by site specific nucleases (e.g., Zinc Finger Nucleases (ZFNs), Meganucleases, Transcription Activator-Like Effector Nucelases (TALENS) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease (CRISPR/Cas) with an engineered crRNA/tracr RNA), to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination of an exogenous donor DNA polynucleotide, such as a transgene, within a predetermined genomic locus. This predetermined genomic locus is not obvious. Many sites in the genome are non-ideal for, for example, transgene insertion, due to highly repetitive nucleotide sequence, methylation, and other characteristics that result in a very high or very low level of recombination or poor expression of genes on introduced transgenes. Therefore, there is a need in the art to identify ideal target sites within a genome for targeted modifications, such as transgene insertion.
Once a target site has been used for targeted modification, there is a need to determine if the desired targeted modification was successfully created. Existing methods of screening for targeted genomic modifications in cells are primarily based on polymerase chain reaction (PCR) protocols, nucleic acid sequencing and Southern analysis. In the case of PCR amplification, the screening process of handling the complexity of gene insertion or modification at a specific site is inefficient due to the complexity of PCR primer settings and inherent ambiguity of PCR amplification due to the resulting complexity of genome rearrangement and genome ploidy. Some of the problems with PCR include: 1) no clear distinction between one copy and two copy insertions due to ploidy of the genome; 2) a requirement for complex primer design and large sets of primer combinations to deal with the complexity of gene insertion or modification at the specific site(s); and 3) low throughput of gel electrophoresis and ambiguity of amplification bands. Although subsequent sequencing can help in identifying the characteristics of PCR amplification products, there are problems with large scale sequencing efforts and interpretation of results for large sample numbers. Further gene segregation analysis is required to isolate homozygous progeny for further screening. These steps require large scale operations for screening of commercial crops in order to capture less than 2% of potential candidates and the inventory scale of plants in greenhouses require commercial scales of space and operational costs until the plant growth stage is mature enough to carry out Southern analyses.
The present invention addresses these shortcomings in the art by providing an ideal target site for a maize genome. The present invention also provides a more strategic and efficient approach to identify and enrich for cells with a targeted genomic insertion or a targeted genomic mutation, which reduces the number of candidate plants with high accuracy at the very early stages of the screening process, avoiding a large scale sequencing effort and reducing greenhouse operational costs for plant maintenance.